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Detachabead

Manufactured by Thermo Fisher Scientific
Sourced in United States

DETACHaBEAD is a magnetic bead-based cell separation product used for the isolation and purification of specific cell types from complex biological samples. It enables the effective separation and recovery of target cells without the need for complex instrumentation.

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19 protocols using detachabead

1

Isolation of CD19+ B cells from healthy human donors

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Buffy coats of healthy human donors were obtained from Sanquin Blood Supply. All the healthy donors provided written informed consent in accordance with the protocol of the local institutional review board, the Medical Ethics Committee of Sanquin Blood Supply, and the study conformed to the principles of the Declaration of Helsinki. Peripheral blood mononucleated cells (PBMCs) were isolated from buffy coats using a Lymphoprep (Axis-Shield PoC AS, Dundee DD2 1XA, Scotland) density gradient. Afterwards, CD19+ B cells were isolated using magnetic Dynabeads (Invitrogen) and DETACHaBEAD (Invitrogen) according to the manufacturer’s instructions.
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2

Isolation of High-Purity CD8+ T Cells

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CD8+ T cells were positively isolated from PBMCs and detached from beads by an immunomagnetic method (CD8 Positive Isolation Kit; Dynabeads® plus DETACHaBEAD®; Invitrogen) according to the manufacturer’s instructions. The resulting purity was >99%, and the viability was >95%.
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3

Isolation of Human Blood Cells

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Human buffy coats were obtained from healthy blood donors after informed consent, in accordance with the protocol of the local institutional review board, the Medical Ethics Committee of Sanquin Blood Supply, and conforms to the principles of the Declaration of Helsinki. Peripheral blood mononuclear cells (PBMCs) were isolated through standard gradient centrifugation using Ficoll-lymphoprep (Axis-Shield). CD19+ B cells and CD4+ T cells were purified from PBMCs with anti-CD19 and anti-CD4 Dynabeads, respectively, and DETACHaBEAD (Invitrogen) following the manufacturer's instructions. Purity was typically > 98% as assessed by flow cytometry.
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4

Isolation of CD19+ B-cells from Buffy Coats

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Buffy coats were collected from voluntary, non-remunerated, adult healthy blood donors (Sanquin Blood Supply, Amsterdam, the Netherlands), who provided written informed consent for the use of remainders of their donation for research as part of routine donor selection and blood collection procedures. Peripheral blood mononucleated cells (PBMCs) were isolated from buffy coats using a Lymphoprep (Axis-Shield PoC AS, Dundee, Scotland) density gradient. Afterward, CD19+ B-cells were separated using magnetic anti-CD19 Dynabeads and DETACHaBEAD (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions with purity >99%.
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5

Isolation of CD19+ B-cells from Buffy Coats

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Buffy coats were collected from voluntary, non‐remunerated, adult healthy blood donors (Sanquin Blood Supply, Amsterdam, the Netherlands), who provided written informed consent for the use of remainders of their donation for research as part of routine donor selection and blood collection procedures. Peripheral blood mononucleated cells (PBMCs) were isolated from buffy coats using a Lymphoprep (Axis‐Shield PoC AS, Dundee, Scotland) density gradient. Afterward, CD19+ B‐cells were separated using magnetic anti‐CD19 Dynabeads and DETACHaBEAD (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions with purity > 99%.
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6

CD19+ B Cell Isolation and EBV Transformation

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CD19+ B cells were isolated from PBMCs using Dynabeads CD19+ pan B (11143D, Invitrogen) according to the manufacturer’s instructions. 2.5 × 108 cells of PBMCs were resuspended in 10 ml isolation buffer (PBS, 0.1% BSA, 2 mM EDTA). 250 μl of prewashed beads were added to PBMCs and incubated for 20 min in 4°C with gentle rotation. For positive isolation of CD19+ B cells, beads and supernatant were separated using magnet, and supernatant was discarded. Beads were washed three times, and beads bounded with CD19+ B cells were resuspended with 2.5 ml of cell culture medium (80% RPMI 1640, 20% heat-inactivated FBS, glutamine). CD19+ B cells were released from Dynabeads using DETACHaBEAD (Invitrogen, ca12506D) according to the manufacturer’s instruction.
B cells were infected with EBV to transform lymphocytes. 10 ml of B cells were transferred into a T75 flask. 1.5 ml of stock EBV collected from a B95-8 strain-containing marmoset cell line and 1 ml of phytohemagglutinin P (PHA-P) were added to flask and incubated in 37°C with a 5% CO2 atmosphere incubator. Every 5–7 d, 10 ml of cell culture medium was added. Cells were let to grow in the CO2 atmosphere incubator for 30 d until all B cells were transformed to LCLs.
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7

Culturing B-cell and Monocytic Lines

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Two human B-cell lines (GM12878 and Raji (GM04671) cells, both from Coriell Institute) and a monocytic cell line, U937 (ATCC CRL-1593.2), were cultured in RPMI 1640+GlutaMAX-I medium (Gibco), 1% penicillin-streptomycin (Sigma-Aldrich) and complement-inactivated 10% FBS (Gibco) at +37 °C and 5% CO2.
Primary tonsillar B cells from two seronegative individuals of the study cohort were isolated with anti-CD19 magnetic beads from frozen total cell suspensions of mechanically homogenized preparations and released with DETACHaBEAD as instructed by manufacturer (Invitrogen).
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8

Isolation and Activation of B Cells

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Blood was withdrawn from 3 HLA-A2 positive healthy volunteers (Sanquin), PBMCs were isolated by Ficoll gradient separation. Monocytes were isolated through plastic-adherence by incubating fresh PBMCs in IMDM ++ for 1 h at 37°C and 5% CO2 on culture plastic. B cells were isolated using anti-CD19 Dynabeads and DETACHaBEAD (Invitrogen) according to manufacturer's protocol. B cell were activated by incubating them with 0.1 µM CpG (Invivogen) and 2.5 µg/mL Fab mix (anti-IGA, IgG and IgM, Jackson ImmunoResearch) in IMDM ++ for 3 days at 37°C and 5% CO2.
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9

Induction of colitis via T-cell transfer

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Colitis was induced by AdTr of CD4+CD25 T cells (AdTr-colitis) from spleen of MHC-compatible BALB/c mice to C.B-17 SCID recipients as previously described [9 (link)]. Briefly, splenocytes of BALB/c donor mice were subjected for positive selection of CD4+ T cells using Dynabeads and DETACHaBEAD (Life Technologies Europe, Ballerup, Denmark) and depletion of CD25+ from the CD4+ T cell suspensions using the CD25 MicroBead kit. Flow cytometry was used to analyze purity of the cells and showed that more than 98% of the CD4+ cells were CD25 cells. Each recipient was reconstituted with 300,000 cells by intraperitoneal injection. Two or three weeks after transfer, peripheral blood from all mice was analyzed by flow cytometry for the presence of CD4+ T cells. Only animals with successful transplantation of cells were included in the study.
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10

B cell immortalization from apheresis

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B cells were isolated from apheresis cones (NHS Blood and Transplant) by positive selection with CD19 Dynabeads (Life Technologies) followed by removal of the beads with Detachabead (Life Technologies). B cells were then infected with recombinant 2089 EBV at 100 MOI as previously described (Shannon-Lowe et al., 2005 (link)) and cells harvested at specific time points, washed, pelleted and frozen at −80 °C.
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